FIG. 5.

Genetic requirements for EsxB and EsxW secretion. Wild-type and isogenic mutants esxB, esxW, esxL, and essC were grown to an A_{600 nm} of 3 in LB medium with glucose (LBG) or LB medium with glucose-sodium bicarbonate (LBG CO₂). One-milliliter cell cultures (Total) or equivalent volumes of culture supernatants (Sup) were precipitated with TCA. Proteins in extracts were solubilized in SDS-PAGE sample buffer prior to separation on 15% acrylamide gels and transfer to a polyvinylidene difluoride membrane for immunoblotting with specific antibodies (anti-EsxB, anti-EsxW, and anti-L6 as a loading control). Immunoreactive signals were revealed using a secondary antibody coupled to horseradish peroxidase and chemiluminescence and captured on film. The intensity of bands corresponding to EsxB and EsxW immune species was measured by scanning the films. All samples were compared to the wild-type total extract set as 100%. α, anti; WT, wild type.